The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein represents the N-terminal domain of the HIV-1 precursor Gag (PrGag) protein and carries an N-terminal myristate (Myr) group. HIV-1 MA fosters PrGag membrane binding, as well as assembly of envelope (Env) proteins into virus particles, and recent studies have shown that HIV-1 MA preferentially directs virus assembly at plasma membrane sites enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2). To characterize the membrane binding of MA and PrGag proteins, we have examined how Myr-MA proteins, and proteins composed of Myr-MA and its neighbor Gag capsid (CA) protein associate on membranes containing cholesterol and PI[4,5]P2. Our results indicate that Myr-MA assembles as a hexamer of trimers on such membranes, and imply that MA trimers interconnect CA hexamer rings in immature virus particles. Our observations suggest a model for the organization of PrGag proteins, and for MA-Env protein interactions.

SummaryDuring morphogenesis of mature HIV-1 cores, the viral capsid (CA) proteins assemble conical or tubular shells around the viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which CA proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle pre-existing tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature HIV-1 core assembly, and avenues for antiviral inhibition.

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 Å is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers. During human immunodeficiency virus 1 (HIV-1)1 assembly, the viral precursor Gag (PrGag) protein oligomerizes on cellular membranes and directs the budding of immature virus particles. Normally, during or after budding, PrGag proteins are processed into the mature Gag proteins: matrix (MA), capsid, nucleocapsid (NC), and p6 (1). PrGag processing is accompanied by a dramatic change in HIV-1 particle morphology. By conventional thin section electron microscopy, the electrondense protein shell of immature virions appears to reorganize into centrally located, conical, or cylindrical mature virus cores (1).Recently, we demonstrated that the major HIV-1 Gag protein capsid could assemble in vitro into two distinct arrangements (2). Both of these arrangements showed hexameric rings of capsid N-terminal domains (NTDs) linked via C-terminal domain (CTD) contacts but differed in that one showed tight packing of hexamers with adjacent NTD rings in apparent contact, whereas the other featured clearly separated NTD rings (2). Interestingly, the two in vitro arrangements appear to have in vivo counterparts. The tightly packed arrangement is similar to that observed in immature virions, where hexamer-to-hexamer spacing is in the range of 65 to 80 Å (2-5). In contrast, the loosely packed arrangement appears to correspond to the organization of capsid proteins in mature cores assembled in vitro and in vivo with a hexamer ring-to-ring spacing of 95-110 Å (6, 7). The similarity between our results and those observed in vivo prompted us to propose a model in which viral morphogenesis was accompanied by a shift from tightly packed hexamer rings to loosely packed rings, and we predicted that virions would have mo...